If the described method of microplate alamar blue biofilm susceptibility testing, which is simple, reproducible, cost. Franzblaumicroplate alamar blue assay versus bactec 460 system for highthroughput screening of compounds against mycobacterium tuberculosis and mycobacterium avium antimicrob. Microplate alamar blue assay for staphylococcus epidermidis biofilm susceptibility testing. The main obstacle in antimycobacterial discovery is the extremely slow growth rates of pathogenic mycobacteria that lead to the long incubation times needed in antimycobacterial screening.
Original article resazurin microtiter assay for detection of. Some in vitro testings has been developed and are currently available for antimycobacterial screening. Adjust the cell count to 1 x 10 4 cellsml suggested cell density. Dec 30, 20 another brothbased method, the microplate alamar blue assay, has been used for years with favorable results for mycobacterium tuberculosis complex isolates. Alamar blue redox dye is a type of resazurin that enters live, viable cells and then becomes reduced intracellularly. The 96well microplate alamar blue assay maba allows for the quantitative determination of drug susceptibility against any strain of replicating mycobacterium tuberculosis to be completed within a week at minimal cost. A simple method to measure cell viability in proliferation. Pdf microplate alamarblue assay for paracoccidioides. Long hansens disease center, baton rouge, louisiana 70894, usa. Simple resazurinbased microplate assay for measuring. The inexpensive microplate alamar blue assay maba is an indirect colorimetric dst method for determining the mics of tb drugs for strains of mycobacterium tuberculosis. Here we describe a stepbystep procedure for both the maba and lora. Microplate alamar blue assay versus bactec 460 system for. A colorimetric, microplate based alamar blue assay maba method was used to determine the mics of isoniazid inh, rifampin, streptomycin sm, and ethambutol emb for 34 peruvian mycobacterium tuberculosis isolates including both pansensitive and multidrugresistant strains and the h37rv strain by using bacterial suspensions prepared directly from solid media.
This is a trusted and established reagent which has been available since 1993. The rema plate method has proven to be, in recent experience, a reliable method for the detection of mdr tb 23. The aim of the study was to compare resazurin microplate assay rema and crystal violet decolorization. The microplate alamarblue assay maba is a sensitive, rapid, inexpensive, and nonradiometric method, which evaluates metabolic function and cellular health and offers the potential for screening large numbers of antimicrobial compounds. The alamarblue hs and alamarblue cell viability reagents are readytouse resazurinbased reagents that function as cell health indicators by using the reducing power.
When compared with gold standards such as bactec 460. Validation of the alamarblue assay as a fast screening. A simple method to measure cell viability in proliferation and cytotoxicity assays 256 braz oral res 2009. Pdf the use of the microplate alamar blue assay maba to. Compared to alamarblue, alamarblue hs contains highly purified resazurin and provides higher sensitivity, and a larger assay window. Biofilms from the tested candida albicans strains were markedly resistant to amphotericin b amb, nystatin nyt, fluconazole flc and 5fluorouracil 5fc, but susceptible to. For example, ab results can be read visually, spectrofluorometrically or spectrophotometrically, its reduction is dependent on active metabolism, it is amenable to highthroughput, its use. Stock solutions of all extracts at a concentration of 20 mgml were freshly prepared on the same day of the s1maba by resuspending 200 mg powder of each plant extract in 10 ml of sterile mq water. The inexpensive microplate alamar blue assay maba is an indirect colorimetric dst method for determining the mics of tb drugs for strains of mycobacterium tuberculosis 10. In the present study, a resazurin microplate method rmm using the fluorescent redox dye resazurin was developed. Horton5 cancer research institute,1 department of chemistry and biochemistry,2 and school of life sciences,3. The results were compared to those of the proportion method pm on lj. If the described method of microplate alamar blue biofilm.
This method used alamar blue, a resazurinbased oxidationreduction based indicator. The alamarblue assay is designed to measure quantitatively the proliferation of various human and animal. L collins and s g franzblau pharmacology research department, gillis w. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Microplate based alamar blue assay maba is a practical, sensitive and inexpensive assay method of cell viability in which fluorescence reduction assay of alamar blue, a resazurin a dark blue dye and nonfluoroscent in oxidized form, available as a sterile and liquid reagent commercially, is used in a microplate format.
Microplate alamar blue assay for susceptibility testing of candida. Alamar blues stability and lack of toxicity allowed cfuml to be determined from the same wells as alamar blue absorbances. The resazurin assay also known as alamar blue assay offers a simple, rapid, and sensitive measurement for the viability of mammalian cells and bacteria. Method for measuring cytotoxicity or proliferation using alamarblue by fluorescence harvest cells which are in the log phase of growth and determine cell count. The bioassay may also be used to establish relative cytotoxicity of agents within various chemical classes 3. Microplate reader assays for cell proliferation biocompare. A colorimetric, microplatebased alamar blue assay maba method was used to determine the mics of isoniazid inh, rifampin, streptomycin sm, and. Cell lines are often cultivated in contact with the target materials, and after a variable period. Microplate alamar blue assay maba and low oxygen recovery assay lora for. Trek diagnostic systems, in comparison with these other methods of susceptibility testing, may have many potential advantages. A simple method for the isolation and purification of total lipides from animal tissues.
Microplate alamarblue assay maba for the determination of antimicrobial activity of each plant extract, mabas were performed 19, 23, 24. Resazurin is blue and nonfluorescent whereas resorufin is red and highly fluorescent. It is a sensitive assay if working with higher than 5x10 3 cells per 100. We investigated a highthroughput method for determining the susceptibility of candida albicans biofilms that uses the oxidation reduction indicator alamar blue ab. The optimum cell density may vary between cell types. Alamarblue assay for cell proliferation bmg labtech. Antimicrobial susceptibility of standard strains of. Resazurin changes from blue nonfluorescent to pinkhighlyfluorescent testing format. If the described method of microplate alamar blue biofilm susceptibility testing, which is simple, reproducible, costeffective, nontoxic, and amenable to high throughput, is applicable to other. Microplate alamar blue assay maba and low oxygen recovery. Tetrazolium microplate assay as a rapid and inexpensive.
The use of the microplate alamar blue assay maba to assess the. Alamar blue s stability and lack of toxicity allowed cfuml to be determined from the same wells as alamar blue absorbances. Citeseerx microplate alamar blue assay for staphylococcus. Calcein am provides a simple, rapid, and accurate method to measure cell viability andor cytotoxicity. Resazurin microtiter assay plate method for detection of. The alamarblue hs and alamarblue cell viability reagents are readytouse resazurinbased reagents that function as cell health indicators by using the reducing power of living cells to quantitatively measure viability.
We investigated a highthroughput method for determining the susceptibility of candida albicans biofilms that uses the. A similar reagent, known as water soluble tetrazolium salts wsts, are tetrazolium dyes that do not enter cells but become reduced extracellularly by. Resazurin 7hydroxy3hphenoxazin3one 10oxide is a phenoxazine dye that is weakly fluorescent, nontoxic, cellpermeable, and redox. In vitro antimycobacterial activity of six cameroonian. Another brothbased method, the microplate alamar blue assay, has been used for years with favorable results for mycobacterium tuberculosis complex isolates. In this study, we used the microplate alamar blue assay to test the activities of 15 drugs against 24 standard ntm strains in china. The subsequent cell signals can be detected using microplate readers. Inter and intraassay reproducibility of microplate alamar. The concentration of resazurin measured by the cqs was strongly correlated with the values measured by the elisa microplate reader y 206. Resazurin has been identified as the main component of alamar blue 22. Moreover, coban 2012 reported 100% agreement between the resazurin microplate assay for the rapid determination of mrsa and the cefoxitin mic determined by the reference broth microdilution method.
Antituberculosis activity of brotowali tinospora crispa. Microplate alamar blue assay for susceptibility testing of. Comparison between the cqs method and microplate reader quantification. As examples, there are those that monitor dna synthesis by the use of either a radioactive label 3 hthymidine uptake or immunofluorescence antibrdu monoclonal antibody. Microplate alamar blue assay for staphylococcus epidermidis. Microplate alamar blue assay for staphylococcus epidermidis bio. Antimycobacterialactivity tests the antimycobacterial activity of the plant crude extracts was tested using the microplate alamarblue assay 18,19.
Throughput in tuberculosis drug discovery was extremely limited prior to the introduction of microplate based susceptibility assays. L, relatively nontoxic and provided that it is carried out carefully, the same replicates can be followed over several time points. Measuring cytotoxicity or proliferation alamarblue assay. Since alamar blue has been recently identified as resazurin in cell cytotoxicity studies, we have standardized and evaluated a microplate method which uses the reduction of resazurin for dst to inh and rif in clinical isolates of m. With a pure culture, this method can determine antibiotic susceptibility in 6 to 7 days. International journal of mycobacteriology4 2015 306311 307. Maba is a microplate method that used as highthroughput screen antituberculosis activity of samples. The 96well microplate alamar blue assay maba allows for the quantitative. Pettit r, weber ca, kean mj, hoffmann h, pettit g, tan r et al. This method, the microplate alamar blue assay maba, is based on the detection of colorimetric changes caused by the oxidation and reduction capabilities of alamar blue dye.
1247 1324 363 943 292 412 670 252 728 473 1017 661 1275 1300 1276 318 107 1069 991 1492 500 288 771 421 238 1304 61 118 1137 391 898 1152 1111 119 910 205